Pertinhez T A, Nakaie C R, Paiva A C, Schreier S
Departamento de Bioquímica, Universidade de São Paulo, Brazil.
Biopolymers. 1997 Dec;42(7):821-9. doi: 10.1002/(SICI)1097-0282(199712)42:7%3C821::AID-BIP7%3E3.0.CO;2-V.
A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.
合成了一种自旋标记的十五肽，其N端氨基酸为2,2,6,6 - 四甲基哌啶 - N - 氧基 - 4 - 氨基 - 4 - 羧酸（TOAC），并包含大量癌基因受体的253 - 266位残基（EYWSTFGNLHHISL），该受体是G蛋白偶联受体家族的一种膜结合蛋白。据预测，这种蛋白质折叠成由三个细胞外环和三个细胞内环连接的七个跨膜螺旋，而该肽包含第三个细胞外环的一部分和第七个螺旋的一部分。自旋标记肽（TOAC - 14）的电子顺磁共振（EPR）光谱是在水溶液中作为pH和温度的函数获得的，在一种诱导二级结构的溶剂[三氟乙醇（TFE）]中，以及在去污剂胶束和磷脂双层存在的情况下获得的。TOAC和TOAC - 14的带电荷和不带电荷的氨基产生了具有不同各向同性超精细分裂（aN）的光谱。在EPR时间尺度上质子化和非质子化形式之间的缓慢交换产生了由亨德森 - 哈塞尔巴尔赫方程加权的复合光谱。aN对pH的作图允许确定氨基的pK值（TOAC和TOAC - 14分别为8.4和4.5）。在pH 6.5处aN的微小变化归因于组氨酸的滴定。计算得到的旋转相关时间值表明存在pH诱导的构象变化。在TFE中也观察到了构象变化。TOAC - 14与胶束结合，与肽和去污剂头部基团的电荷无关。相反，该肽仅在两者带有相反电荷时才与磷脂双层结合。膜结合的和游离的TOAC - 14之间的缓慢交换（在EPR时间尺度上）允许计算肽的分配系数。光谱线形状受聚集体大小和组成分子的堆积程度影响。有人提出，pH、极性和脂质环境可以影响膜结合受体水暴露区域的构象，从而在信号转导机制中发挥作用。
